Glucocorticoids are small lipophilic compounds that mediate their many biological effects by binding an intracellular receptor (GR) that, in turn, translocates to the nucleus and directly or indirectly regulates gene transcription. Perhaps the most recognized biologic effect of glucocorticoids on peripheral T cells is immunosuppression, which is due to inhibition of expression of a wide variety of activationinduced gene products. Glucocorticoids have also been implicated in Th lineage development (favoring the generation of Th2 cells) and, by virtue of their downregulation of fasL expression, the inhibition of activation-induced T cell apoptosis. Glucocorticoids are also potent inducers of apoptosis, and even glucocorticoid concentrations achieved during a stress response can cause the death of CD4(+)CD8(+ )thymocytes. Perhaps surprisingly, thymic epithelial cells produce glucocorticoids, and based upon in vitro and in vivo studies of T cell development it has been proposed that these locally produced glucocorticoids participate in antigen-specific thymocyte development by inhibiting activation-induced gene transcription and thus increasing the TCR signaling thresholds required to promote positive and negative selection. It is anticipated that studies in animals with tissue-specific GR-deficiency will further elucide how glucocorticoids affect T cell development and function.

Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding of [14C]iodoacetamide-labeled calponin to actin. The labeling procedure did not significantly alter the binding constant of calponin to actin. The stoichiometry of binding was variable and dependent on ionic strength. Below 110 mM ionic strength, the stoichiometry of binding was 1:1. As the ionic strength was increased above 110 mM ionic strength, the stoichiometry shifted from 1:1 to 1 calponin per 2 actin monomers. At physiological ionic strength, the binding exhibited a small degree of positive cooperativity and was adequately described by a single class of binding sites with an association constant of 6 x 10(6) M-1. The affinity decreased to 20% of this value in the presence of ATP. Irrespective of the ionic strength, actin formed bundles when saturation with calponin exceeded about 30%. Measurements of the rate of association were complicated by this bundling, but the upper limit for this reaction was placed at 10(6) M-1 s-1. The addition of calponin to actin-caldesmon complexes caused displacement of the caldesmon.

We have proposed that glucocorticoids antagonize TCR-mediated activation and influence which TCR avidities result in positive or negative selection. We now analyze the immune response of mice whose thymocytes express antisense transcripts to the glucocorticoid receptor (TKO mice). TKO mice responded normally to the complex antigen PPD but were proliferative nonresponders to pigeon cytochrome c 81-104 (PCC), having a large decrease in the frequency of PCC-responsive CD4+ T cells. Unlike wild-type T cells, few TKO T cells in PCC-specific cell lines expressed V alpha11+Vbeta3+. Furthermore, for naive CD4+ T cells from unimmunized TKO mice, the frequencies of many of the molecular features common to the CDR3 regions of PCC-responsive V alpha11+Vbeta3+ cells were substantially decreased. Thus, thymocyte glucocorticoid hyporesponsiveness resulted in loss of cells capable of responding to PCC, corresponding to an antigen-specific "hole" in the T cell repertoire.